Flow cytometer is an instrument for measuring/analyzing individual fluorescently-labeled particles/cells being lined up and flown through a flow channel under hydrodynamic focusing or other focusing forces. Light beam from individual light sources (e.g. laser) of particular wavelengths is shaped typically to an elliptical or rectangular shape with the major/long axis (about 50 to 200 microns) perpendicular to the flowing direction of the particles/cells in a flow channel and minor/short axis (about 10 to 30 microns) parallel to the flowing direction of the particles/cells and is guided/directed to optical interrogation zones (OIZ) in the flow channel. As the fluorescently-labeled particles/cells pass through the light beam one-by-one in the optical interrogation zones, multiple physical characteristics of single cells can be detected and measured. The properties measured include a particle's relative size (Forward Scatter, i.e. FSC), relative granularity or internal complexity (Side Scatter, i.e. SSC), and relative fluorescence intensity (i.e., fluorescence signals from fluorescent molecules in the labeled cells under excitation by light sources.). These characteristics are determined using an optical-to-electronic coupling system (i.e. photodetector) that records how the cell or particle scatters incident laser light and emits fluorescence.
Traditionally, two approaches have been implemented for flow cytometers with multiple-excitation light sources. In the first approach, the shaped elliptical laser beams from multiple sources (e.g., S1, S2 in FIG. 1) of different wavelengths propagate across the flow channel and form two optical interrogation zones (OIZ) at different vertical locations along the flow channel, spaced at certain distance in the range of, e.g., 100 to 200 microns. Thus when a cell flows through the flow channel, it will pass the individual OIZ (e.g. OIZ1 corresponding to laser beam S1 and OIZ2 corresponding to laser beam S2 in FIG. 1) in sequence. Consequently, light in the laser beams will be scattered and fluorescent light will be emitted as well by different fluorescent molecules (FM) possessed by the cell. Multiple fluorescent signals (FS) with different peak wavelengths may be emitted by different molecules at one OIZ. For example, S1 can be 488 nm laser and S2 can be 640 nm laser. Fluorescent molecules FITC, PE, and PE-Cy7 can be excited by S1 (488 nm) and emit light at peak wavelength of 519 nm, 578 nm, and 785 nm respectively whilst APC and APC-Cy7 can be excited by S2 (640 nm) and emit light at peak wavelength of 660 nm and 785 nm respectively. It is possible that the spectra of the fluorescent signals excited by different light sources are overlapped (e.g. PE-Cy7 and APC-Cy7 are both emitted at a peak wavelength of 785 nm, but need to be excited by different wavelengths). Therefore, it would be essential that the fluorescent signals excited by different light sources (e.g. FS1 being excited by S1 and FS2 being excited by S2 in FIG. 1) are separated by the light collection and separation optics for effective detection of these fluorescent signals. Otherwise, the flexibility of choosing the fluorochrome for cell staining would be limited and it becomes a shortcoming for a flow cytometer. Through light collection/separation optics, the fluorescent signal FS1 and FS2 is collected and separated to different physical positions with a spatial distance large enough to accommodate the filters and photodetectors for light splitting, filtering, and detection. This light splitting, filtering, and detection system resolves the quantities of each corresponding fluorescent molecule, thus are referred to fluorescence (FL) channels in flow cytometry. For example, fluorescent signal FS1 emitted from OIZ1 is then further split and detected by photodetector D1 and D2 at two different emission wavelengths to resolve the quantity of fluorescent molecule FM1 (e.g., FITC) and FM2 (e.g., PE-Cy7) respectively and fluorescent signal FS2 emitted from OIZ2 is then further split and detected by photodetector D3 and D4 at two different emission wavelengths to resolve the quantity of fluorescent molecule FM3 (e.g., APC) and FM4 (e.g., APC-Cy7). Photodetectors convert the detected fluorescent signals into electronic signals. Using electronic processors, such electronic signals are then filtered, amplified and converted into digital signals, and further processed to derive various characteristics of each corresponding fluorescent molecule. For example, output E1 and E2 correspond to the signals of FM1 and FM2 respectively and output E3 and E4 correspond to the signals of FM3 and FM4 respectively. When a particle/cell passes through an OIZ, an electronic pulse will be generated at the corresponding detection channel and such cell/particle induced electronic pulse is characterized by its height, area, and width to reveal the property of the particles/cells. For example, the height of the pulse detected by one FL channel provides a good indication of the intensity of the corresponding fluorescent molecule. In summary, in a flow cytometer with a system configured shown in FIG. 1 and as described above, fluorescent signals excited by different light sources is physically/optically separated for detection with different photodetectors, and fluorescent molecules with overlapped emission spectra could be used to label the particles/cells in the same experiment.
However, the first approach has several limitations. (1) Complex optics system are needed to collect and to separate light from different OIZs in order to achieve an efficient and clear separation of light emitted from each OIZ with small physical separation distances of about 100-200 microns. (2) Accurate control and delivery of excitation beams to the flow channel is required to have an accurate control of separation distance between OIZs. In a flow cytometer, an individual cell is usually transported through the flow channel at a constant speed, which means that the time interval between the detected digital pulse generated in OIZ1 and the detected digital pulse generated in OIZ2 will be fixed. Therefore, in order to correlate signals from the same cell passing through different OIZs, this time interval should be controlled accurately as well. Furthermore, light emitted from each OIZ is separated out from each other using a collection and separation optics system and detected by a different set of filters and photodetectors. The efficiency of light collected and delivered to photodetectors for each OIZ depends on the separation distances between OIZs. If the separation distance between these OIZs varies due to any factors such as temperature, pressure, misalignment during instrument shipping, or other system instability factors, it may affect not only the time interval between detected pulses generated in two OIZs but also, more importantly, the efficiency of light collection for each OIZ, leading to unreliable measurement results of the signals. (3) The optical setup is not efficient because each photodetector is used to detect only one type of fluorescent molecule (i.e. having a pre-determined excitation wavelength and emission bandwidth). For example, in a system as illustrated in FIG. 1, four detection channels (E1 to E4) are needed to record the signals from four fluorescent molecules (FM1 to FM4). If FM2 (e.g. PE-Cy7) and FM4 (e.g. APC-Cy7) are assumed to have the same/overlapped emission spectrum but need to be excited by S1 and S2 respectively, two sets of band-pass filters and photodetectors are still needed for effective detection. Combining of the detection of FM2 and FM4 with one band-pass filter and one photodetector is not possible in this configuration. This results in the increased complexity and therefore increased cost of the whole system.
In the second approach, the shaped, elliptical laser beams from multiple sources (e.g., S1, S2 in FIG. 2) of different wavelengths propagate across the flow channel at a single vertical location along the flow channel, forming a single optical interrogation zone (OIZ). Thus when a particle/cell flows through the flow channel, it will pass the OIZ and will be subjected to multiple laser beams simultaneously. Consequently, light in laser beams will be scattered and fluorescent light will be emitted by fluorescent molecules (FM) possessed by the cell. Multiple fluorescent signals with same/overlapped or different emission peak wavelengths may be emitted by different fluorescent molecules excited by multiple laser beams at the OIZ. For example, S1 and S2 can be 488 nm and 640 nm laser, respectively. Fluorescent molecules FITC and PE-Cy7 can be excited by S1 (488 nm) and emit light at peak wavelength of 519 nm and 785 nm, respectively, whilst fluorescent molecules APC and APC-Cy7 can be excited by S2 (640 nm) and emit light at peak wavelength of 660 nm and 785 respectively. Through light collection optics, the fluorescent signals emitted from the particle/cell in the OIZ would be collected, and then further split or filtered into different wavelength ranges and is detected by photodetectors, converting optical signals into electronic signals. For example, fluorescent signals emitted from OIZ is split and detected by D1, D2 and D3 at three different emission wavelengths to resolve quantity of fluorescent molecules FM1 (e.g., FITC) and FM2 (e.g., PE-Cy7 or PE-Cy7) and FM3 (APC). Using electronic processors, electronic signals are then filtered, amplified and converted to digital signals, and further processed to derive various characteristics of each corresponding fluorescent molecule. For example, output E1, E2 and E3 correspond to the signals of FM1, FM2 and FM3, respectively. This second approach, as schematically represented in FIG. 2, has some advantages. There is no need for complex optical system for separating fluorescent signals excited by multiple light sources. Furthermore, the optical setup in this approach is efficient since the same set of optical filter and photodetector could be used for detection of fluorescent molecules having the same emission peak wavelengths but different excitation wavelengths. For example, whilst PE-Cy7 and APC-Cy7 are excited by 488 nm and 640 nm respectively, both molecules can be detected using the same set of band-pass filter and one photodetector for monitoring wavelength ranges centered at 785 nm, as long as these two dyes are not used in the same experiment. On the other hand, this approach has a major limitation that it could not distinguish the fluorescent signals from different fluorescent molecules having the same emission spectra even if they are excited by different lasers. For example, the system of such a configuration as schematically shown in FIG. 2 could not be used to distinguish and reliably detect fluorescent signals from molecules of PE-Cy7 (excited by 488 nm laser) and APC-Cy7 (excited by 640 nm laser) in the same experiment, even though they have different excitation wavelengths.
A relatively recent method, published in U.S. Pat. No. 7,990,525, describes an extension of this second approach where the excitation laser light is time-multiplexed so that each light source is switched on and off at a very fast rate. At any time moment, no two (or more) light sources are switched on simultaneously. Thus, either no light source or only one light source is switched on by appropriate control of light sources. The detection electronics can be used in synchronization so that the fluorescence signals excited by different lasers could be isolated, recovered, and analyzed. It is required that the multiple excitation light beams are directed/focused to the same OIZ, and consequently there is no time interval between detected electronic signals excited by different excitation light sources as seen for the first approach. However, this approach possessed other limitations. Firstly, in order to eliminate the above-mentioned time interval issue, it requires precise combination and alignment of the light beams from different excitation light sources to be coaxial and overlapped at the same location across the flow channel. Secondly, for reliable recovery and isolation of the emitted fluorescent signals excited by different excitation light sources as a particle/cell passes through the OIZ, accurate control of the time-multiplexed illumination of multiple excitation light beams is essential as well as the subsequent synchronization of the signal processing electronics. Thirdly, the time-multiplexed illumination of multiple excitation light sources corresponds to a fact that the time interval for a cell/particle to pass through the OIZ is shared between multiple excitations. Consequently, a single particle-induced electronic pulse generated when the particle/cell passes through the OIZ is time-shared between multiple excitations as well. If the same amount of data points is needed to effectively recover such particle-induced electronic pulse information for each of the multiple excitations, a faster multiplexing illumination rate and a faster sampling frequency for the signal processing electronics is required. Furthermore, such issue would become more severe especially when the number of the excitation light sources increases. Fourthly, such configuration requires that one excitation light source is OFF when another one is ON. However, due to stray current of the electronic signal for the digital modulation of the laser source and/or the property of the laser source (i.e. modulation ratio is not high enough so that there is still low level of light from the light source even when it is controlled to be OFF), such OFF-status light source will still contribute some illumination to the OIZ thus increase the background for detection and measurement of the emitted fluorescent signal excited by another ON-status light source, affecting the system sensitivity for detecting low-level, dim fluorescent particles/cells.
Another recent method, published in U.S. Pat. No. 8,077,310, also describes a further extension of the previously described second approach where two excitation sources emit lights at different wavelengths onto a single location on a flow channel. The multiple excitation sources are controlled to operate between such operational modes: a first mode wherein only one of multiple excitation sources emits light onto the single location and a second mode wherein both excitation sources emit lights onto the single location. The approach further comprises a detector subsystem that detects lights emitted from the single location and generates a composite signal and a processor to separate the composite signal into component signals due to each of two excitation sources. Since the multiple excitation light beams are directed to the same single location on the flow channel, consequently there is no time interval between detected electronic signals excited by different excitation sources as seen for the first approach. By switching between different operational modes, composite signals corresponding to these operation modes are generated and can be processed to result in isolated signals due to each individual excitation sources. This approach provides a possibility of, in a single experiment, using different fluorescent molecules having the same emission spectra but with different excitation wavelengths. However, such an approach has limitations due to emitting multiple excitation sources onto a single location on the flow cell and simultaneous turn-on of multiple excitation sources at some time moments.
Still other approaches have been suggested or described in recent years, relating to emitting multiple excitation light sources onto a flow channel and detecting and separating emission fluorescent lights due to these excitation sources. For example, US 2008/0213915 described an approach where multiple excitation light sources are all modulated with each source being modulated at different frequencies. The modulated excitation beams are combined and guided onto single or multiple focused spots or locations on the flow channel. The fluorescent emissions from particles due to modulated excitation beams are detected to produce detector output signals, which are then processed to distinguish the fluorescent signals caused by each individual excitation beam. In another example, US 2007/0096039 described an approach for analyzing objects having multiple fluorescing species in a fluid stream. Multiple intensity-modulated excitation light beams, each of which is modulated at a unique frequency between 2 and 100 MHz, are combined and directed to one or more interrogation zones on a flow cell and will interact with the passing objects in a fluid stream in the flow cell. The fluorescence emission light from fluorescent species in the objects is detected with one or more photosensitive detectors and resulted electronic signals are analyzed to extract multiple component emission signals, each of which corresponds to one excitation light beam. These approaches have limitations associated with the requirement of modulation of all excitation sources at unique frequencies and the ineffectiveness in the de-modulation methods in achieving high signal-noise ratios. Thus, there remains a need to develop a novel approach for effective detection of emission light by multiple excitation light sources from a flow channel.